Ab41803

Cholecalciferol (vitamin D3) (C9756, Sigma Aldrich, St. Louis, MO, USA). Mouse monoclonal antibody against α-SMA (ab7817, Abcam, USA), rabbit polyclonal antibodies against acid ceramidase-α (AC^α) (sc-292176, Santa cruz, USA), RUNX2 (ab23981, Abcam, USA), OSP (ab63856, Abcam, USA), SM22-α (ab14106, Abcam, USA), VPS16 (Cat. No.17776-1-AP, Protein biotech group, USA), Rab7 (ab137029, Abcam, USA), CD63 (ab216130, Abcam, USA), annexin-II (AnX2, ab41803, USA), and alkaline phosphatase (ALP, sc-28904, Santa Cruz, USA). Rat monoclonal anti-mouse Lamp-1 (ab25245, Abcam, USA), Cre (Cat No. 6905, Novagen EMD Millipore, Billerica MA, USA), and ceramide (MID 15B4, Enzo ALX-804-196-T050). Secondary antibodies are Alexa-488 or Alexa-555-labeled (Life technologies, USA). Von Kossa staining kit (ab150687, Abcam, USA) and Alizarin Red S Solution (TMS-008-C, EMD Millipore. USA) were used for detection AMC.

Neutrophils were suspended into DMEM/F12 medium plus 1% FBS at 5 × 10⁶/mL and added into 24-well plate with coverslip coated with poly-D-lysine. The cells were stimulated with 2 μg/mL rA2 for 30 min at 37 °C and washed with PBS. Subsequently, cells were fixed in 4% paraformaldehyde for 15 min and were further incubated with rabbit anti-AXNA2 (Abcam, ab41803) and mouse anti-TLR4 (Santa Cruz, sc-293027) overnight at 4 °C. After washing, cells were incubated with Alexa Fluor 594-labeled donkey anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG for 1 h at 4 °C in the dark. Finally, after mounted on glass slides, fluorescence signals on the cells were visualized by using a Leica TCS SP2 confocal microscope at × 600 magnification.